中兽医医药杂志2025，Vol.44Issue(4)：18-25,8.DOI:10.13823/j.cnki.jtcvm.2025.040
啮齿类实验动物4种常见病原菌多重巢式PCR检测方法的建立与初步应用
Establishment and preliminary application of multiplex nested PCR detection method for four pathogenic bacteria in rodent laboratory animal
摘要
Abstract
The purpose of this study is to establish a more accurate,sensitive,fast and simple multiplex nested PCR method that can detect four common pathogens:Salmonella typhimurium,Corynebacterium kutscheri,Pseudomonas aeruginosa and Staphylococcus aureus in rodent laboratory animal.On this basis,the sampling methods of throat swabs and anal swabs were adopted to reduce the waste of animal resources and optimize the detection process.According to the conservative and variable regions of the 16S rDNA sequence of pathogenic bacteria,universal primers and specific primers were designed,respectively.Through the difference of primer length,pathogenic bacteria in various samples were detected by nested PCR amplification processes.The results showed that the optimized 4-multiplex nested PCR method could specifically amplify the four pathogenic bacteria in the same sample without cross reaction.The multiplex nested PCR could amplify the DNA fragments of the target pathogenic bacteria from the standard strains DNA template,but no fragments were detected in the negative control group.The sensitivity of multiplex nested PCR was significantly higher than that of ordinary multiplex PCR(≤10-2 pg/μL vs≤1 pg/μL).The multiplex nested PCR method could also specifically detect the target pathogens from co-infection disease material.In practical applications,multiplex nested PCR could detect positive samples of pathogenic bacteria infections,and the detection rate was higher than that of ordinary multiplex PCR.The established multiplex nested PCR system can simultaneously detect multiple pathogens in clinical samples such as animal throat swabs or anal swabs.Compared with ordinary multiplex PCR method,this method has the advantages of time-saving,labor-saving,high sensitivity and specificity.关键词
多重巢式PCR/鼠伤寒沙门菌/鼠棒状杆菌/铜绿假单胞菌/金黄色葡萄球菌Key words
multiplex nested PCR/Salmonella typhimurium/Corynebacterium kutscheri/Pseudomonas aeruginosa/Staphylococcus aureus分类
农业科技引用本文复制引用
席晓霞,孙婧,郭家熙,张丽娟,刘晓玲,段天林..啮齿类实验动物4种常见病原菌多重巢式PCR检测方法的建立与初步应用[J].中兽医医药杂志,2025,44(4):18-25,8.基金项目
甘肃省中医药研究中心开放课题(zyzx-2024-zx08) （zyzx-2024-zx08）
甘肃省自然科学基金实验动物专项(23JRRA1169) （23JRRA1169）
